The following discussion of immunoassays and definitions of terms often used with respect to immunoassays are set forth herein as background to facilitate the understanding or the disclosure and claims hereof.
The term "analyte" refers to the protein, which may be, but is not necessarily, an antibody, to be detected.
The term "test sample" typically refers to a sample of body fluid such as plasma, serum, ascites, lymphatic fluids, cerebral spinal fluid, nipple fluid discharge, urine and other body fluids that may contain the analyte of interest. Optionally, the test sample can be diluted in a suitable diluent buffer, such as phosphate buffered saline with serum components, to provide a sample volume that is required by the particular immunoassay.
The term "specific binding member" refers to a member of a specific binding pair, i.e., two different molecules wherein one of the molecules through chemical or physical means specifically binds to the second molecule. In addition to antigen and antibody specific binding pairs such as the allergen and antibody pair, other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an antibody specific for the sequence protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g., ribonuclease, S-peptide and ribonuclease S-protein), and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member, for example an analyte-analog. If the specific binding member is an immunoreactant it can be, for example, an antibody, antigen, hapten, or complex thereof. If an antibody is used, it can be a monoclonal or polyclonal antibody, a recombinant protein or antibody, a mixture or mixtures or a fragment or fragments thereof, as well as a mixture of an antibody and other specific binding members. The details of the preparation of such antibodies and their suitability for use as specific binding members are well-known to those skilled-in-the-art.
The term "indicator reagent" refers to an assay reagent comprising a detectable label directly or indirectly attached to a specific binding member which is capable of directly or indirectly binding to the analyte and thereby indicating the presence, absence or amount of the analyte in a test sample. A variety of different indicator reagents can be formed by varying either the label or the specific binding member. In general, the indicator reagent is detected after it has formed a complex with either the analyte or a complementary specific binding member, but the unbound indicator reagent can also be detected.
The term "label" refers to any substance which is attached to a specific binding member and which is capable of producing a signal that is detectable by visual or instrumental means. Labels can include chromogens; catalysts; fluorescent compounds; chemiluminescent compounds; radioactive isotopes; direct visual labels including colloidal metallic and non-metallic particles, dye particles, enzymes or substrates, or organic polymer latex particles; liposomes or other vesicles containing signal producing substances; and the like.
Many enzymes suitable for use as labels are disclosed in U.S. Pat. No. 4,275,149, columns 19-23, herein incorporated by reference. For example, an enzyme/substrate signal producing system useful with 4-methylumbilliferyl phosphate is the enzyme alkaline phosphatase. If horseradish peroxidase is used, o-Phenylenediamine is added as an enzyme substrate to form a colored product which can be detected and/or measured visually or instrumentally.
In an alternative signal producing system, the label can be a fluorescent compound where no enzymatic manipulation of the label is required to produce a detectable signal. Fluorescent molecules such as fluorescein, coumarin, phycobiliprotein, rhodamine and their derivatives and analogs are suitable for use as labels in this system.
Another class of labels includes the visually detectable, colored particles which enable a direct colored readout of the presence or concentration of the analyte in the test sample without the need for using additional signal producing reagents. Materials for use as such particles include colloidal metals, such as gold, and dye particles as disclosed in U.S. Pat. Nos. 4,313,734 and 4,373,932. The preparation and use of non-metallic colloids, such as colloidal selenium particles, are disclosed in co-owned U.S. Pat. No. 4,954,452, which is incorporated by reference herein. Organic polymer latex particles for use as labels are disclosed in co-owned and copending U.S. patent application Ser. No. 248,858, filed Sep. 23, 1988, now allowed which is incorporated by reference herein. The selection of a particular label is not critical, so long as the label is capable of generating a detectable signal either by itself or in conjunction with one or more additional signal producing substances. The term "signal producing component" refers to any substance capable of reacting with another assay reagent or the analyte to produce a reaction product or signal that indicates the presence of the analyte and that is detectable by visual or instrumental means. "Signal production system", as used herein, refers to the group of assay reagents that are needed to produce the desired reaction product or signal. For example, one or more signal producing components can be used to react with a label and generate the detectable signal, i.e., when the label is an enzyme, amplification of the detectable signal is obtained by reacting the enzyme with one or more substrates or additional enzymes to produce a detectable reaction product. The term "capture binding member" refers to a specific binding member which can bind directly or indirectly to the analyte or indicator reagent and which is bound or is capable of being bound to a solid phase, or is capable of being precipitated, such that the capture binding member can be separated from the test sample and other assay reagents.
The term "capture reagent" refers to a capture binding member which is directly or indirectly attached to a solid phase material to enable the separation of the capture binding member, and analyte or indicator reagent that is bound thereto, from unbound analyte and assay reagents. Typically, the attachment of the capture binding member to the solid phase material is substantially irreversible and can include covalent mechanisms. A capture reagent in which a capture binding member is indirectly attached to a solid phase can be produced by reacting a coupling agent of the instant invention with both the solid phase material and the capture reagent; the product of such a reaction is an example of a "conjugate". In an agglutination assay, the capture binding member of the capture reagent can be bound to a soluble carrier material such as bovine serum albumin.
In producing a capture reagent to be used in an assay, once the capture binding member, e.g., analyte specific antibody, is immobilized upon the solid phase, the remaining surface area of the solid phase is generally blocked with a suitable protein solution, such as bovine serum albumin, to prevent non-specific binding of protein to the support. The solid support is then washed with an appropriate solution to remove any excess blocking solution and/or unbound capture binding member.
Once complex formation occurs between the assay components, the solid phase can be used as a separation mechanism. For example, the reaction mixture can be contacted with the solid phase material, and the solid phase material retains the newly formed reaction complex(es). Alternative methods can be used to perform this separation step, such as using a solid phase which itself binds to the capture binding member; affixing to the solid phase a binding member that is specific for the capture binding member; or affixing to the solid phase a reactive agent, such as a charged substance, which will attract and bind an oppositely charged substance that has been bound to the capture binding member, as disclosed in co-owned and copending U.S. patent application Ser. No. 150,278, filed Jan. 29, 1988, now Ser. No. 816,786, which is incorporated by reference herein. Either the binding member that is specific for the capture binding member or the reactive agent (e.g., a charged substance) can be bound to or chemically reacted with a coupling agent according to the invention which is also bound to or chemically reacted with the solid phase material; these are also examples of conjugates.
Assay devices can have many configurations, several of which are dependent upon the material chosen for the solid phase. The term "solid phase material"=0 refers to any suitable chromatographic, bibulous, porous or capillary material or other conventional solid material, well-known to those skilled-in-the-art for use in immobilizing specific binding members. Solid phase materials can include a fiberglass, cellulose or nylon pad for use in a flow-through assay device having one or more layers containing one or more of the assay reagents; a dipstick for a dip and read assay; a test strip for chromatographic (e.g., paper or glass fiber) or thin layer chromatographic (e.g., nitrocellulose) techniques in which one or all of the reagents are contained in separate zones of a single strip of solid phase material; or an absorbent material well known to those skilled-in-the-art. The solid phase material can also include, without limitation, polyacrylamide beads, polystyrene beads or tubes, magnetic beads, a microtitre plate with one or more reaction wells, microparticles or a glass or plastic test tube. Natural, synthetic or naturally occurring materials that are synthetically modified, can be used as a solid phase material including polysaccharides, e.g., cellulose materials including paper, cellulose and cellulose derivatives such as cellulose acetate, nitrocellulose and cellulose acetate/nitrate; silica; fiberglass; inorganic materials such a deactivated alumina, diatomaceous earth or other inorganic finely divided material uniformly dispersed in a porous polymer matrix, with polymers such as vinyl chloride, vinyl chloride-propylene copolymer, and vinyl chloridevinyl acetate copolymer; cloth, both naturally occurring (e.g., cotton) and synthetic (e.g., nylon); porous gels such as silica gel, agarose, dextran and gelatin; polymeric films such as polyacrylamide; magnetic particles; microtitre plates; polystyrene tubes; protein binding membranes; Sephadex (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.); Trisacryl (Pointet-Girard, France); silicon particles; porous fibrous matrixes; and the like. The solid phase material should have a reasonable inherent strength or strength can be provided by means of a support, and it should not interfere with the production of a detectable signal.
When the specific binding member of the capture reagent is affixed to microparticles, those particles can be retained in a column, suspended in a mixture of soluble reagents and test sample, or retained and immobilized by another solid phase base material. By "retained and immobilized" is meant that the particles, associated with the solid phase base material, are not capable of substantial movement to positions elsewhere within that material. The size of the particles is not critical, although it is preferred that the average diameter be smaller than the average pore size of the solid phase base material if such is used, and they must be of such a size that they can be suspended in a suitable liquid if they are to be used in an agglutination assay.
The term "ancillary specific binding member" refers to a specific binding member used in addition to the capture binding member and the indicator reagent which becomes a part of the detectable binding complex. One or more ancillary specific binding members can be used in an assay. For example, an ancillary specific binding member can be used in an assay where the capture binding member is capable of binding the ancillary specific binding member which is in turn capable of binding the solid phase.
It will be appreciated by those skilled-in-the-art that the selection of any given label, ancillary binding member or solid phase material is generally not critical to the present invention. The materials are chosen to optimize the results provided by the chosen assay configuration.
It has been disclosed, European Patent Application 0 221 505, published May 13, 1987, that immunogens suitable for intravenous administration can be produced by incubation with pepsin immobilized on a hydrophilic gel containing amino groups by a dicarboxylic acid disuccinimidyl ester. The disuccinimidyl ester is disclosed as having a succinimidyl group at each end of a chain having the structure ##STR4## where R is a straight or branched chain alkylene radical having from 1 to 20 carbon atoms. The application specifically discloses only one such compound: adipic acid disuccinimidyl ester where R, above, is a straight chain alkylene group having 4 carbon atoms.
Various bifunctional coupling agents are commercially available, for example from Pierce, exemplary ones being disclosed in a catalog published by that company. Disuccinimidyl suberate and ethylene glycolbis(succinimidylsuccinate) are examples of such coupling agents disclosed in the catalog. In the latter compound, R in the foregoing formula is ##STR5##
Cobalamins have the general structure shown in FIG. 1 of the attached drawings. While cobalamins have sometimes been referred to as vitamin B12, there are actually several different types of cobalamins which differ from each other by the R substituent shown in the FIG. 1 structure: cyanocobalamin (R=cyano), hydroxycobalamin (R=hydroxy), aquacobalamin (R=H.sub.2 O), nitrocobalamin (R=NO2), 5' deoxyadenosylcobalamin (R=5' deoxyadenosyl), and methylcobalamin (R=methyl). Each of these cobalamins is considered generally to be a vitamin B12: cyanocobalamin (vitamin B12), hydroxycobalamin (vitamin B12a), aquacobalamin (vitamin B12b), nitrocobalamin (vitamin B12c), 5' deoxyadenosylcobalamin (coenzyme B12), methylcobalamin (methyl B12). The various cobalamins have similar metabolic activity. Cyanocobalamin, however, is more stable than the others. The cobalamins are involved in many metabolic functions and are essential for normal growth and nutrition, hematopoiesis, production of all epithelial cells, and maintenance of myelin throughout the nervous system.
A deficiency in vitamin B12 manifests itself in ineffective hematopoiesis, inadequate myelin synthesis, inadequate maintenance of the epithelial cells of the alimentary tract, and generalized anemia. However, except for inadequate myelin synthesis, these symptoms are common to many megaloblastic anemias, regardless of cause.
To pinpoint the cause of such anemias, it is necessary to test for vitamin B12 deficiencies. There are a variety of different assays for vitamin B12: colorometric, spectroscopic, fluorometric and radioactive isotope. The most common employs a cobalt 57 radioactive isotope in lieu of the cobalt in the corrin nucleus of the vitamin B12 molecule. The radioactively labelled molecule and B12 intrinsic factor are added to a sample containing B12, and the radioactively labelled B12 and the B12 in the sample compete for binding sites on B12 intrinsic factor. The B12 intrinsic factor is associated with a solid phase, so the amount of radioactivity on the solid phase or in the sample will be proportional to the amount of B12 in the original sample. The current radioassays have obvious disadvantages inasmuch as they involve the handling, storage, and disposal of radioactive materials. Furthermore, detection can be slow.
Enzyme linked competitive binding assays have been proposed (see Bachas, Biotechnics, vol. 4, no. 1, p. 42 et seq. (1986)) for vitamin B12. However, the sensitivity of the assay was reported to be 1355 pg/ml while the normal range for vitamin B12 in human serum is from 150-900 pg/ml. Clearly, such an assay cannot be used to test vitamin B12 deficiency since it reportedly cannot even detect vitamin B12 at normal ranges in human serum.